Therapeutic potentials of combined use of DMSA with calcium and ascorbic acid in the treatment of mild to moderately lead intoxicated mice

The aim of this study was to explore the therapeutic efficacies of combined use of meso-2,3-dimercaptosuccinic acid (DMSA) with calcium and ascorbic acid in the treatment of mild to moderately lead-intoxicated mice. Female albino mice were exposed to lead by drinking water contaminated with 0.1% (moderate lead exposure) or 0.05% (mild lead exposure) lead acetate. After the cessation of lead exposure, mice were supplemented by gavage with saline solution, 50 mg/kg body weight (b.w) DMSA, 100 mg/kg b.w DMSA, calcium and ascorbic acid, or 50 mg/kg b.w DMSA and calcium as well as ascorbic acid, respectively. Atomic absorption spectrophotometric method was used to analyze lead levels in blood, bone, liver, kidney and brain. Activities of blood δ-aminolevulinic acid dehydratase (ALAD) were determined by colorimetric method. DMSA supplemented alone could reduce lead levels in both soft tissues and bone and reverse lead-inhibited activities of blood ALAD in mild to moderately lead-intoxicated mice. On the other hand, combined use of DMSA with calcium and ascorbic acid achieved better therapeutic efficacies in mobilizing lead in blood, liver and kidney, and reversing lead-inhibited activities of blood ALAD in moderately lead intoxicated mice than DMSA supplemented alone. Moreover, the better therapeutic efficacies were also found in mildly lead intoxicated mice in mobilizing lead in blood and bone achieved by combined use of DMSA with calcium and ascorbic acid. Combined use of DMSA with calcium and ascorbic acid seems to be the better choice in the treatment of mild to moderate lead-intoxication.     

Interactions of the chelating agent 2,3-dimercaptopropane-1-sulfonate with red blood cells in vitro. II. effects on metalloproteins

The implications of the carrier mediated uptake of 2,3-dimercaptopropane-1-sulfonate (DMPS) (D.B. Wildenauer et al., Chem.-Biol. Interact., 42 (1982) 165) on cytoplasmic components of human red blood cells have been investigated in vitro. The water-soluble chelating agent caused a mobilization of metals (zinc and copper) from metalloproteins which resulted in a permeation of the membrane. Furthermore, a cytoplasmic protein was found to be attached to the membrane after DMPS treatment of red blood cells. The protein was isolated and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid analysis and finger-printing as carbonic anhydrase. The enzyme could be solubilized from the membrane by addition of β-mercaptoethanol, suggesting an involvement of sulfhydryl-groups. In a reconstitution experiment, DMPS-treated human carbonic anhydrase could be attached to inside-out vesicles which were prepared from human erythrocytes. In contrast, bovine carbonic anhydrase, which is known to lack sulfhydryl-groups, failed to bind to the same vesicles. Moreover, attachment of carbonic anhydrase to the membrane did not occur when intact bovine erythrocytes were treated with DMPS. It is suggested that zinc-depletion of carbonic anhydrase causes the liberation of a sulfhydryl-group of the enzyme. This is followed by a disulfide formation with a component of the membrane which results in the observed membrane attachment.     

Preparation of (S)-2,3-dichloro-1-propanol byPseudomonas sp. and its use in the synthesis of (R)-epichlorohydrin

Summary Pseudomonas sp. OS-K-29 assimilated (R)-2,3-dichloro-1-propanol preferentially as the sole source of carbon. Isolation of optically pure (S)-2,3-dichloro-1-propanol with 100% enantiomer excess (e.e.) from the racemate was done based on this bacterial assimilation using immobilized-cells of OS-K-29 with calcium-alginate. The overall examination of the reactor involved 19 batches for 50 days without loss of its activity. Highly pure (R)-epichlorohydrin with 99.5% e.e. was prepared from the (S)-2,3-dichloro-1-propanol with treatment of aqueous NaOH. This new method is simple and useful for manufacturing optically active (S)-2,3-dichloro-1-propanol and (R)-epichlorohydrin.     

Interactions of the chelating agent 2,3-dimercapto-propane-1-sulfonate with red blood cells in vitro. I. Evidence for carrier mediated transport

Uptake of the water soluble 1,2-dimercaptopropanol (BAL) derivative 2,3-dimercapto-1-sulfonate (DMPS) into human red blood cells was found in vitro and the mode of penetration studied in detail. The compound entered erythrocytes in a concentration dependent manner. In contrast to sealed ghosts where inside and outside concentrations reached the same value, DMPS accumulated in intact erythrocytes. Since no binding of DMPS could be detected, the reason for accumulation was assumed to be a conversion of DMPS into chelates or metabolites which penetrated the membrane in a slower rate. A facilitated transport of DMPS mediated by the anion carrier protein was concluded on the basis of the following similarities with the anion transport: inhibition of [¹⁴C]DMPS-uptake by N-ethylmaleimide (NEM), tetrathionate (90%), sulfate (50%), 5,5′-dithio bis(2-nitrobenzoic acid) (DTNB) (25%); inhibition of uptake and efflux by 4,4′-diisothiocyano-2,2′-stilbene disulfonate (DIDS) (80%), dipyridamole (55%); temperature dependency (activation energy 24 K_cal/mol); pH-dependency (pH optimum about 6.9); counter-transport; activation of uptake by preincubation with DMPS (transmembrane effect).     

Preparation of DL-2,3,4-trihydroxybutylarsonic acid and dl-2,3-dihydroxybutane-1,4-bis(arsonic acid): starting compounds for novel arsonolipids

The reaction of DL-1,3-butadiene diepoxide and of DL-1,4-dibromo-2,3-butanediol with aqueous alkaline sodium arsenite, "Na(3)AsO(3)", gave mixtures of the title arsonic acids which can be separated by anion exchange resin. Characterization of by-products leads to a better understanding of these reactions. These compounds are valuable intermediates for the preparation of novel arsonic acids and bis(arsonic acids).     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

A reinvestigation of the synthesis of arsonolipids (2,3-diacyloxypropylarsonic acids)

A reinvestigation of the reactions leading to arsonolipids (2,3-diacyloxypropylarsonic acids) has been carried out in order to understand why the yields of their preparation were only moderate, although they are better than those reported for 2,3-diacyloxypropylphosphonic acid (phosphotidic acid). Thus, the reaction of glycidol and of 3-chloro-1,2-propanediol with alkaline sodium arsenite, "Na3AsO3", gives the desired product, 2,3-dihydroxypropylarsonic acid, and approximately 10% of an arsenic-containing glycerol dimer which is removed during the preparation of these arsonolipids. The step which is mainly responsible for the diminished yields is due to the reaction of the -As(SPh)2 or -AsO3H- precursor with the activated acid chlorides or carboxylic acid anhydrides to give an intermediate which cyclizes with the primary hydroxy group of the 2,3-dihydroxypropyl moiety. This cyclization does not allow the primary hydroxy group to be acylated. Such cyclization could not be avoided with RCOCl/py, (RCO)2O/DMAP, or RCOOH/DCC/DMAP acylating systems.     

Biosynthesis of enantiopure (S)-3-hydroxybutyric acid in metabolically engineered Escherichia coli

A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the beta-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl beta-D: -thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the beta-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l(-1) from 20 g l(-1) of glucose in Luria-Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l(-1) (OD(600) = 30 and 60), respectively. When protein expression was induced at 60 of OD(600) and glucose was fed to the concentration of 15 g l(-1), 10.3 g l(-1) of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l(-1)h(-1). Lowering glucose concentration to 5 g l(-1) and induction of protein expression at 30 of OD(600) significantly reduced final S3HB concentration to 3.7 g l(-1), which also resulted in the decrease of the S3HB productivity to 0.05 g l(-1)h(-1).     

An autoantibody against N-(carboxyethyl)lysine (CEL): Possible involvement in the removal of CEL-modified proteins by macrophages

Advanced glycation end products (AGEs) are believed to play a significant role in the development of diabetic complications. In this study, we measured the levels of autoantibodies against several AGE structures in healthy human plasma and investigated the physiological role of the autoantibodies. A high titer of the autoantibody against N^ε-(carboxyethyl)lysine (CEL) was detected in human plasma compared with other AGE structures such as CML and pentosidine. The purified human anti-CEL autoantibody reacted with CEL-modified human serum albumin (CEL-HSA), but not CML-HSA. A rabbit polyclonal anti-CEL antibody, used as a model autoantibody against CEL, accelerated the uptake of CEL-HSA by macrophages, but did not enhance the uptake of native HSA. Furthermore, when ¹²⁵I-labeled CEL-HSA was injected into the tail vein of mice, accumulation of ¹²⁵I-CEL-HSA in the liver was accelerated by co-injection of the rabbit anti-CEL antibody. These results demonstrate that the autoantibody against CEL in plasma may play a role in the macrophage uptake of CEL-modified proteins.     

Isolation and identification of (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid from anAmanita of the sectionRoanokenses (Amanitaceae)

A chlorine-containing non-protein amino acid which was recently discovered from the fruit bodies ofAmanita gymnopus (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid, was isolated and crystallized for the first time from the fruit bodies of an unknown member ofAmanita belonging to the sectionRoanokenses, subsectionSolitariae. The results of elementary analyses, determination of optical rotations,¹H- and¹³C-NMR-spectra, and some chemical reactions supported an earlier proposed structure.Part 24 in the series Biochemical studies of nitrogen compounds in fungi. for Part 23, see Hatanaka, S. I. et al. 1994. this journal35: 391–394.     

Model systems for the immunolocalisation of cis,trans abscisic acid in plant tissues

To explore the feasibility of immunolocalisation of endogenous abscisic acid (ABA), model systems were developed for testing quantitatively the sensitivity of the second antibody peroxidase/antiperoxidse (PAP) method for immunolocalisation of ABA on plant tissues. Exogenous (±)ABA was fixed to carrot sections on glass slides or to homogenised pea cotyledon material on microtitre plates, either directly by carbodiimide fixation or by glutaraldehyde fixation of ABA-protein conjugates linked through the C1 carboxyl by 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride (EDC). Backgrounds were decreased by including 0.1% normal goat serum in the incubations, by including 0.1% Triton X-100 as a wetter, by including glycine in the rinses after EDC fixation and by using low-pH rinses after incubation with the primary antibody. Serum antibodies recognising the peptide bond between the protein and abscisic acid were removed by preincubating the serum with acetic acid conjugated to protein. Positives were only accepted when they could be eliminated by adding an excess of ABA-protein conjugate in the primary antiserum. By using a soluble peroxidase reaction product to facilitate quantitation, the limit of reliable exogenous ABA detection was found to be only of the order of 1 pmol. For the histochemical immunolocalisation of endogenous ABA, better antisera and lower backgrounds will be required.The efficiency of fixation of exogenous ABA was determined using [³H] or [¹⁴C]ABA. When aqueous EDC or di-isopropyl carbodiimide (IPC) were used the fixation efficiency was low (up to 5%), but much higher efficiencies (up to 80%) were obtained using IPC vapour with freeze-dried material. Similarly efficient fixation of endogenous ABA in pea cotyledon material, as determined by gas chromatography-mass spectrometry analysis, was obtained using the same technique. The PAP method failed to detect fixed endogenous ABA in pea cotyledons, even though the total tissue amounts present exceeded 1 pmol, evidence that not enough of the ABA was accessible to the antibody.Abbreviations ABA abscisic acid - ACE-ALP acetic acid-alkaline phosphatase - EDC 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride - GC-MS gas chromatographymass spectrometry - IgG Immunoglobulin G - HSA humanserum albumin - IPC dinsopropyl carbodiimide - LINK goat anti-rabbit IgG - OD optical density - PAP peroxidase/rabbit antiperoxidase complex     

Production of l-lactic acid by electrodialysis fermentation (EDF)

An efficient process for producing l-lactic acid using an, EDF method is described. The results showed that intermittent EDF with continuous medium feed was the best one among the experiment methods employed. Comparing with the conventional EDF, intermittent EDF (seven on–off) with continuous medium feed indicated that the maximum value of o.d.₆₆₀ was not increased, but productivity was 1.5 times higher. The yield increased by above 30% and glucose transport decreased to 1/10 (from 0.46 to 0.05).     

Production of poly(3-hydroxybutyric-co-3-hydroxyvaleric acid) having a high hydroxyvalerate content with valeric acid feeding

The capability of different organic acids to produce a derivative of PHB [poly(3-hydroxybutyric-co-3-hydroxyvaleric acid), P(3HB-co-3HV)] was examined in shake flask cultivations. Propionic and valeric acids demonstrated the potential to produce P(3HB-co-3HV) under nitrogen limiting conditions at 30°C. The addition time and the initial concentration of valeric acid needed for a high cellular HV content were identified by extensive experimentation. Fed-batch cultivation in 7-l bioreactor with valeric acid feeding resulted in the production of PHA containing 54% HV units.     

Use of the enriched stable isotope Cr-50 as a tracer to study the metabolism of chromium (III) in normal and diabetic rats

The activable enriched stable isotope Cr-50 compound Cr₂O₃ was used as a tracer to study the metabolism of chromium(III) [CR(III)] intragastrically administered in normal and diabetic rats. The comparison of absorption, distribution, and excretion in organs and tissues of the two groups do not show much alteration, but some differences exist indeed. The contents of⁵¹Cr radioactivity of the diabetic rats appear to be of higher retention than in most studied organisms. The urinary⁵¹Cr excretion of diabetics is significantly higher than that of normal rats. Therefore, a conclusion can be drawn that the insulin-dependent rats generally absorb and excrete more chromium (Cr) than the normal rats.     

Application of hexafluoroacetone as protecting and activating reagent in amino acid and peptide chemistry

Summary Using hexafluoroacetone as protecting and activating reagent, multifunctional amino acids like aspartic acid can be functionalized regioselectively. This strategy offers i.a. a two-step synthesis for aspartame and preparatively simple access to multifunctional natural and unnatural amino acids, like 4-oxo-L-amino acids, 5-diazo-4-oxo-L-amino acids, 4-substituted L-proline derivatives and various heterocyclic L-amino acids. On application of this strategy to amino diacetic acid N-substituted glycines become readily available.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 M solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 M, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 M) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 M and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3–100 M unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-¹⁴C]IAA (external concentration 0.2 M). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3–100 M, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.Abbreviations IAA indol-3yl-acetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid     

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

Summary A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.     

First total syntheses of (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid

The first total syntheses for the (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid were accomplished in seven steps and in 31-32% overall yields. The (trimethylsilyl)acetylene was the key reagent in both syntheses. It is proposed that the best synthetic strategy towards monounsaturated iso methyl-branched fatty acids with double bonds close to the omega end of the acyl chain is first acetylide coupling of (trimethylsilyl)acetylene to a long-chain bifunctional bromoalkane followed by a second acetylide coupling to a short-chain iso bromoalkane, since higher yields are thus obtained. Spectral data is also presented for the first time for these two unusual fatty acids with potential as biomarkers and as topoisomerase I inhibitors.